Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

TY - JOUR

T1 - Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

AU - Fogg, Paul C M

AU - Younger, Ellen

AU - Fernando, Booshini D

AU - Khaleel, Thanafez

AU - Stark, W Marshall

AU - Smith, Margaret C M

N1 - © The Author(s) 2017.

PY - 2018/2/16

Y1 - 2018/2/16

N2 - To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase.

AB - To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase.

KW - Journal Article

KW - Attachment Sites, Microbiological

KW - Lysogeny

KW - Integrases/chemistry

KW - Recombinant Proteins/chemistry

KW - Thermodynamics

KW - Viral Proteins/chemistry

KW - Cloning, Molecular

KW - Protein Interaction Domains and Motifs

KW - DNA, Bacterial/chemistry

KW - Binding Sites

KW - Genetic Vectors/chemistry

KW - Streptomyces/chemistry

KW - Amino Acid Sequence

KW - Protein Conformation, alpha-Helical

KW - Gene Expression

KW - Models, Molecular

KW - DNA-Binding Proteins/chemistry

KW - Escherichia coli/genetics

KW - Sequence Homology, Amino Acid

KW - Sequence Alignment

KW - Protein Conformation, beta-Strand

KW - Siphoviridae/chemistry

KW - Protein Binding

KW - Mutation

KW - Amino Acid Substitution

UR - http://www.scopus.com/inward/record.url?scp=85044670915&partnerID=8YFLogxK

U2 - 10.1093/nar/gkx1233

DO - 10.1093/nar/gkx1233

M3 - Article

C2 - 29228292

SN - 0305-1048

VL - 46

SP - 1308

EP - 1320

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 3

ER -